The broad long-term objectives of my research is to understand the molecular basis for target cell killing and immune regulation by natural killer (NK) cells. NK cells provide early defense against infections and tumors. A growing number of recent studies indicate that NK cells also play a critical role in the regulation of adaptive immune responses by its interaction with other lymphocytes and dendritic cells and secreting cytokines. It is now well accepted that NK cell killing depends on a delicate balance of signals received through the inhibitory and activating receptors upon interaction with their ligands. Most of the inhibitory receptors recognize MHC class I molecules whereas the ligands for most of the activating receptors are not well characterized. Among the activation receptors, the natural cytotoxicity receptors (NCRs), including NKp46, NKp44 and NKp30 are NK- specific and play a critical role in the regulation of natural cytolytic function. Despite their molecular characterization and functional studies, the cellular ligands for these receptors remain elusive. In this proposal, I will identify and clone the ligand for NKp44. We have generated a fusion protein containing the extracellular domain of NKp44 fused with the Fc region of human IgG1 and identified a cell line that express the ligand for NKp44. Using the mammalian expression cloning, we will clone the ligand using the NKp44-Fc. COS-7 cells will be transfected with a cDNA library constructed in mammalian expression vector using mRNA from cells that express the ligand. Using NKp44-Fc protein COS-7 cells expressing the NKp44 Ligand will be selected. From these positive cells plasmid DNA will be isolated, sequenced and confirmed by heterologous expression and flow cytometry using NKp44-Fc fusion protein and FITC-conjugated anti- human IgG1. We will also investigate the effect of NKp44-NKp44 Ligand interaction on the functional activity of NK cells. By identifying a target cell that express the NKp44Ligand, and with our proven expertise in molecular biology, we are in a unique position to successfully complete this project. Identification of the ligand for NKp44 will contribute to the understanding of the mechanism underlying natural cytotoxicity by NK cells. This may lead to the development of new immune based therapies for treatment of cancer, immune diseases and infectious diseases. Natural killer cells are the first line of defense against cancer and infections by various pathogens. Here by identifying the ligand for natural cytotoxicity receptor, we will unravel the molecular mechanism by which NK cell kill target cells. This information will be useful in developing immune based treatment for cancer and other diseases. [unreadable] [unreadable] [unreadable] [unreadable]